One of the major challenges in neuroscience is to understand the experience-dependent mechanisms that drive the changes in neural circuits that underlie complex behaviors, and how these mechanisms are altered in disease. Altered synaptic transmission has been implicated in a number of human neurological and psychiatric disorders, including epilepsy, schizophrenia, autism and addiction. Considerable recent evidence from our labs and others has demonstrated that specific patterns of neural activity at individual synapses can drive the growth, stabilization and elimination of synaptic connections. However, how complex patterns of neural activity at multiple synapses in vivo interact to drive changes in circuit connectivity remains poorly defined. Specifically, the relative role of neural activity at clustered versus distributed synaptic inputs, and that of integrated versus patterned neural activity, in driving synaptic and circuit plasticity has been difficult to determine, primarily due to the lack of adequate imaging probes to monitor the history of activity at individual synapses. New tools are needed. The overall objective of this research proposal is to develop novel glutamate sensors for large-scale monitoring of the activity of individual synapses in the living behaving animal. We will focus on two specific objectives: (1) developing glutamate integrators for visualizing the history of neural activity at individual synapses in large fields of view during short behavioral epochs and (2) developing glutamate highlighters with slower kinetics that will enable large-scale monitoring of transient responses at individual synapses in the living animal. Existing tools for monitoring glutamatergic signaling at individual synapses, such as the genetically- encoded glutamate sensor, iGluSnFR, are excellent for characterizing the activity of individual synapses in small fields of view with high temporal precision; however, due to the fast kinetics and transient nature of the glutamatergic responses at individual synapses, these sensors are not suitable for real-time monitoring of synapses in large fields of view. Here, we propose to develop glutamate sensors that permit monitoring activity at individual synapses over larger fields of view and also with the spatial and temporal resolution to mark activated synapses amongst the distributed circuitry. Indeed, our proposed glutamate integrators will transform the activity of transient synaptic inputs into permanent labels of active synapses, enabling access to information mapping neural activity to the structure of the neural circuitry underlying a specific physiological process or behavioral task. To accomplish these goals, we will use a multidisciplinary approach incorporating genetic strategies, computation-guided protein design, two-photon imaging, glutamate uncaging and electrophysiology. First, we will use high-throughput, multi- step sensor screening, photophysical characterization, and ligand-binding specificity measurements to generate candidate sensors. Next, we will generate synaptically targeted sensors, and characterize the sensitivity and kinetics of lead sensors in dissociated neuronal culture and in brain slices. Finally, we will characterize the expression, sensitivity and kinetics of lead sensors in vivo in zebrafish and in mice. If successful, the proposed research will provide much needed imaging tools of synaptic activity that are compatible with a variety of advanced imaging techniques, such as wide-field, confocal and two-photon microscopy, and would dramatically enhance our understanding of how the history of neural activity at individual synapses and their neighbors can influence long-term stability of neural circuit connections.